Despite of rapid advances in elucidating the molecular basis of human diseases, an ostensibly more difficult post-genomic challenge is the functional annotation of disease-specific signaling pathways and the integration of this information into the development of novel drugs. Recent studies demonstrate that CRISPR/CAS9 gene knock-out technology can be used as a powerful tool for large-scale functional genomic analysis in mammalian cells. Unfortunately, although the CRISPR/CAS9 system has opened many potential avenues for improving the drug discovery process, these avenues remain only potential opportunities until we develop robust commercially available CRISPR/CAS9 screening technologies, as well as experimental and bioinformatics tools for data validation and integration of this information into operational cell-based models. The ultimate goal of the proposed project is to develop a novel free public resource lentiviral sgRNA screening platform, enabling scientists to perform genome-wide CRISPR/CAS9 genetic screens in a pooled format. Specifically, we propose using Phase I studies to prioritize the genetic screening performance of 12 different sgRNA designs, and to employ the best validated design to develop a 55K human sgRNA library targeting ~6,500 human genes. Furthermore, we will scale-up the development and commercialization of a comprehensive set of human and mouse genome-wide sgRNA libraries in Phase II studies. These libraries will have improved performance and be designed for cost-effective pooled-format screening and identification of effectors by high-throughput (HT) sequencing. As supporting tools, we will develop protocols, reagents and software tools for screening data analysis and validation. To test the performance of our functional genomics platform, we propose to validate and compare our novel sgRNA and previously established shRNA resource to delineate the processes that underlie tumorigenesis in a panel of PDX-derived lung cancer cell lines. These newly developed sgRNA screening, validation and software tools will be freely available for academic researchers through the open resource www.decipherproject.net site, which was previously funded by NIH and developed by Cellecta for distributing genome-wide shRNA libraries. CRISPR/CAS9 screening products and custom services will provide the research community with highly modular, cost-effective approaches for studies aimed at understanding and integrating dynamic changes in signal transduction networks and ultimately delineating disease-specific phenotypes. The proposed sgRNA screening and bioinformatics strategies harbor considerable potential to systematically identify new targets for therapeutic intervention and to facilitate the development of highly specific drugs, biomarkers and novel therapeutic concepts.